The site of synthesis and the location of the Moloney sarcoma virus (MSV) specific P60 polyprotein in subcellular fractions was determined in pulse-chase experiments. These studies showed that the P60 is synthesized in a low speed sedimenting membrane fraction and becomes associated with the nuclear fraction in a high salt releasable fashion. The latter is consistent with the P60 nucleic acid binding properties. The low speed sedimenting membrane polyribosomes, actively producing this polyprotein were isolated and the mRNA species purified by poly U-sepharose column chromatography. Cell-free translating systems using the homologous or heterologous wheat germ protein synthesizing factors were used to translate the P60 from subcellular fractions or mRNA from polyribosomes. The polyprotein was identified using anti-P60 serum and polyacrylamide gel analysis.